Figure 1.

DKK3 mRNA expression and DKK3 promoter methylation analysis in breast cell lines. (a) DKK3 mRNA expression is differentially expressed in breast cell lines. Non-cancerous cell lines (HMEC and MCF12A) and cancerous breast cell lines (Hs578T, SKBR3, BT20, MCF7, MDA-MB231, T47D and ZR75-1) were analysed by realtime PCR and related to DKK3 mRNA expression in human placental tissue (set to 1). Grey bars = samples expressing abundant DKK3 mRNA; black bars = samples showing strong downregulation of DKK3 expression. (b) Hypermethylation of the DKK3 promoter in breast cell lines. Methylation specific PCR (MSP) was performed with bisulphite-treated DNA from benign and malignant breast cell lines. DNA bands in lanes labelled with U indicate PCR products amplified with primers recognising unmethylated DKK3 promoter sequence. DNA bands in lanes labelled with M represent amplificate generated with methylation-specific primers; water served as 'no template control' (NTC). MSP controls demonstrate the specificity of the DKK3 primers used. Universal poly-methylated bisulphite-converted DNA (UMD) exclusively yields amplification products with primers specific to methylated DKK3 promoter sequence; universal unmethylated bisulphite-converted DNA (UUD) yields exclusive amplification products with primers recognising the unmethylated DKK3 promoter sequence.